The mitochondrion is the organule of the eukaryotic cell responsible for the energy supply. The enzymatic machinery responsible in the last step of the synthesis of the cellular ATP into the H(+)-ATP synthase, a rotary molecular engine that is located in the inner membrane of the mitochondrion [12]. In the last years, the mitochondrion has come to take an important role in studies related to Biomedicine due to its obvious implication in multiple manifestations of the human pathology. Specifically, in the case of cancer, because is the subcellular organule sensor and executor of the apoptosis program. The apoptosis is a genetic program of programmed cell death, that requires energy and is essential for the normal development of the organism. Alterations in the apoptosis program contribute to the progression of certain pathologies such as cancer and neurodegenerative diseases [10, 11].
The study of the energetic metabolism of cancer cells was a central issue in the cancer investigation until the beginning of 1970, decade in which was a relegated topic by the scientific community, due to the approach and development of the molecular biology of this disease. However, it was in 1930 when Otto Warburg formulated the hypothesis that tumour cells should have an altered mitochondrial function and this dysfunction had to explain the high aerobic glycolysis that characterize the majority of the tumour cells. While the glycolytic phenotype of many cancer cells and tumours have been demonstrated from the biochemical and molecular study, dysfunction of the mitochondrial function of the cancer cell was never established in the biology of cancer. In fact, we still are ignorant of the role that the mitochondrion plays in the neoplasic transformation, as well as in the support or promotion of the transformed state of the cell. Solely, and in the case of highly glycolytic rat hepatomas, his aberrant energetic metabolism has been explained by the decrease of the relative cell content of mitochondria [6]. In this concrete case, it has been described that the mitochondrial phenotype of the hepatomas is similar to the mitochondrial phenotype of the fetal liver, where an specific program of mitochondrial biogenesis limits the mitochondrial content of the cell [1].
On the other hand, it has recently been revealed that the execution of the apoptosis program requires the molecular components of the H(+)-ATP synthase of the mitochondrion [7, 8], as well as a functional oxidative phosphorylation in the cell [2, 4]. These results suggest that cells with a relatively low expression of H(+)-ATP synthase and/or with a deficient functionality of oxidative phosphorylation, of which is a bottleneck the same H(+)-ATP synthase, must be more resistant to the apoptosis, and therefore, should have favoured his clonal expansion in the context of the organism.
In the case of the present invention, there have been used monoclonal and polyclonal antibodies that specifically recognize mitochondrial proteins and proteins of the glycolytic pathway in “Western blot” and immunocytochemical techniques, with the aim to quantify the expression of these proteins in normal tissues and its corresponding tumours, to use them, alone in combination, as markers of the progression and prognosis of cancer.
In this way, it has been attained to show for the first time, that the catalytic subunit β of the mitochondrial complex of the H(+)-ATP synthase (β-F1-ATPase) has his expression decreased in liver, kidney and colon tumours, what implies to show that the mitochondrial function of the tumour cell is affected, confirming the hypothesis that Otto Warburg had stated in the beginning of the last century, and that had not been demonstrated in human carcinomas up to this moment. These results allow his extrapolation to mamma, lung, stomach, prostate and endometrium adenocarcinomas; to lung and larynx squamous carcinomas, as well as melanomas and lymphomas.
In liver carcinomas, the affection of the mitochondrial function is produced by repression of the mitochondrial biogenesis, as in a parallel way it is produced the lower expression of structural components of the mitochondrion, as are the marker Hsp 60 and the proper mitochondrial DNA (FIG. 1).
On the contrary, in kidney carcinomas (FIG. 2) and colon carcinomas (FIG. 3), the lower expression of the catalytic subunit β of the mitochondrial complex of the H(+)-ATP synthase is produced in absence of significative changes in the expression of Hsp 60, what indicates that in this type of carcinomas it is being affected the molecular mechanism that determines the grade of functional differentiation of the mitochondrion, that comes expressed by the ratio between the bioenergetic marker and the structural marker, that is, by the proportion β-F1-ATPase/Hsp 60. In mammal adenocarcinomas the lower expression of β-F1-ATPase is produced in a simultaneous way with a higher expression of Hsp 60 and cytochrome oxidase I, what means a very pronounced decrease of the proportion β-F1-ATPase/Hsp 60 and β-F1-ATPase/COX I in the tumour tissue.
In lung adenocarcinomas, the relative expression of β-F1-ATPase decreases very strongly with respect to the expression of the respiratory enzymes cytochrome oxidase I and IV (β-F1-ATPase/COX I and β-F1-ATPase/COX IV).
In lung squamous carcinomas, the relative expression of β-F1-ATPase decreases significantly with respect to the Hsp 60 expression (β-F1-ATPase/Hsp 60).
Moreover, we have shown that in a parallel manner that a decrease in the ratio β-F1-ATPase/Hsp 60, β-F1-ATPase/COX I and/or β-F1-ATPase/COX IV in cancer is produced, it is produced an increase in the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FIGS. 2 and 3) and/or the pyruvate kinase isoform M (PK), markers of the glycolytic pathway.
Because of this reason, we define a bioenergetic index of the cell (BEC index) that relates the bioenergetic potentiality of the mitochondrion with the cellular glycolytic capacity, an index that expresses any of the proportions β-F1-ATPase/Hsp 60, β-F1-ATPase/COX I and/or β-F1-ATPase/COX IV with respect to the cellular expression of GAPDH (ratio β-F1-ATPase/Hsp 60/GAPDH, β-F1-ATPase/COX I/GAPDH, β-F1-ATPase/COX IV/GAPDH and/or PK (ratios β-F1-ATPase/Hsp 60/PK, β-F1-ATPase/COX I/PK, β-F1-ATPase/COX IV/PK). The BEC index also decreases in tumour cells with respect to the observed in healthy cells of the kidney (FIG. 2), colon (FIG. 3), mamma, stomach and lung.
Moreover, in the case of colon carcinomas, the β-F1-ATPase (FIG. 4) as well as the BEC index, are indicators of the patient survival.